Addition of endothelial progenitor cells to renal revascularization restores medullary tubular oxygen consumption in swine renal artery stenosis

Abstract

Renal artery stenosis (RAS) promotes microvascular rarefaction and fibrogenesis, which may eventuate in irreversible kidney injury. We have shown that percutaneous transluminal renal angioplasty (PTRA) or endothelial progenitor cells (EPC) improve renal cortical hemodynamics and function in the poststenotic kidney. The renal medulla is particularly sensitive to hypoxia, yet little is known about reversibility of medullary injury on restoration of renal blood flow. This study was designed to test the hypothesis that PTRA, with or without adjunct EPC delivery to the stenotic kidney, may improve medullary remodeling and tubular function. RAS was induced in 21 pigs using implantation of irritant coils, while another group served as normal controls (n = 7 each). Two RAS groups were then treated 6 wk later with PTRA or both PTRA and EPC. Four weeks later, medullary hemodynamics, microvascular architecture, and oxygen-dependent tubular function of the stenotic kidneys were examined using multidetector computed tomography, microcomputed tomography, and blood oxygenation level-dependent MRI, respectively. Medullary protein expression of vascular endothelial growth factor, endothelial nitric oxide synthase, hypoxia-inducible factor-1α, and NAD(P)H oxidase p47 were determined. All RAS groups showed decreased medullary vascular density and blood flow. However, in RAS+PTRA+EPC animals, EPC were engrafted in tubular structures, oxygen-dependent tubular function was normalized, and fibrosis attenuated, despite elevated expression of hypoxia-inducible factor-1α and sustained downregulation of vascular endothelial growth factor. In conclusion, EPC delivery, in addition to PTRA, restores medullary oxygen-dependent tubular function, despite impaired medullary blood and oxygen supply. These results support further development of cell-based therapy as an adjunct to revascularization of RAS.

Fig. 1.

A: CM-DiI-labeled endothelial progenitor cells (EPC) engrafted in medullary tubular structure (red), shown with 4,6-diamidino-2-phenylindole (nuclear stain, blue) and cytokeratin (tubular marker, green) costains. B: change in medullary blood oxygenation index, R2*, in response to furosemide. BOLD, blood oxygenation level dependent; RAS, renal artery stenosis; PTRA, percutaneous transluminal renal angioplasty. Values are means ± SE. P < 0.05 vs. *sham, †RAS+PTRA+EPC, and #RAS+PTRA.

Fig. 2.

A: microcomputed tomography images of renal microvessels in sham, RAS, RAS+PTRA, and RAS+PTRA+EPC. The vascular density was estimated in outer strip of medulla (highlighted) in all groups. Medullary densities of vessels with diameters smaller than 100 μm were decreased in all groups compared with sham (B), as was capillary density estimated from histology (C). Values are means ± SE. *P < 0.05 vs. sham.

Fig. 3.

Representative (two bands per group) immunoblots demonstrating protein expression of VEGF, endothelial nitric oxide synthase (eNOS), and P47 in the cortex (A) and VEGF, eNOS, hypoxia-inducible factor-1α (HIF-1α), P47, and TNF-α in medulla (B). Values are means ± SE. *P < 0.05 vs. sham.

Fig. 4.

Medullary staining of inducible nitric oxide synthase iNOS (A; top) as an index of inflammation and trichrome (A; bottom) of tubulointerstitial fibrosis (×40), and the quantifications (B). Values are means ± SE. *P < 0.05 vs. sham. †P < 0.05 vs. RAS+PTRA+EPC.